Regenerative cell therapy of the nervous system is an emerging promising mode of therapy. Pluripotent stem cells such as human embryonic stem cells (hESCs) or induced pluripotent stem (iPS) cells may serve as an unlimited source of neurons for transplantation into the nervous system. However, transplantation of pluripotent stem cells-derived neurons may be complicated by the presence of contaminating undesired cells within the transplanted neuronal population. These undesired (contaminating) cells may give rise to the formation of teratoma tumors, and tumors comprised of proliferating neural precursors/progenitors (NPs). In addition, the undesired cells may be the origin of undesired, non-neuronal, cells within the grafts such as neural precursors, non-neural cells or tissues, as well as various glial cells such as astrocytes, microglia or oligodendroglial cells.
Human adult neural stem cells (NSCs) or progenitor cells derived from the brain of aborted fetuses or post natal brain at any age may serve as a source of neurons for transplantation therapy. Nevertheless, transplantation of neurons derived from such cells may also be complicated by the presence of contaminating cells within the transplanted neuronal population. These contaminating cells may also give rise to the formation of tumors comprised of proliferating NSCs or neural precursors/progenitors (NPs)[1], and to the presence of unwanted non-neuronal cells within the graft such as NSCs & NPs, as well as various glial cells such as astrocytes, microglia or oligodendroglial cells.
Parkinson's disease is one example of a condition that may be treated with cell therapy. However, complications that can arise due to the presence of unwanted cells within neuron transplants. Specifically, cell transplantation of dopaminergic (DA) neurons is an attractive therapeutic approach for Parkinsonism, aiming towards restoration of the DA innervations in the affected striatum. Transplantation of fetal mesencephalic tissue in humans showed improvements in some patients. However limited availability of fetal tissue and ethical issues stress the need for alternatives. hESCs may serve as an inexhaustible resource for DA neurons. Several groups have developed and perfected various protocols for derivation of DA neurons from pluripotent stem cells[2][3-5]. Yet, teratoma and neural tumor formation by pluripotent cells and proliferative NPs was demonstrated after transplantation of hESC-derived progeny into animal models of Parkinson's disease[6,7]. These hurdles impede any prospective clinical use of such cells.
Several strategies have been employed so far to avoid teratoma, neural tumor formation, and the existence of unwanted cells within transplanted cell population including, negative and positive selection methods using fluorescence-activated cell sorting (FACS), magnet activated cell sorting (MACS), and immuno-panning all of which require labeling of cell-surface markers for live cell selection. However, specific cell surface markers for labeling desired cell types may not be available or known. For example, in the case of selecting DA neurons for transplantation in Parkinson's disease, a DA neuron-specific cell surface marker has not been identified yet.
To overcome the above problem, genetically modified cell lines have been used. For example, cells expressing green fluorescent protein (GFP) or antibiotics resistance driven by tyrosine hydroxylase (TH)[3] or PitX3[8] promoters that enable antibiotics selection or FACS sorting of transgene-expressing cells were developed for the selection of DA neurons for transplantation in animal models of Parkinson's disease.
Another strategy to enrich for a desired neuronal subtype, which is also based on genetic modification, is forced expression of transcription factors which have a key role during embryonic development of the desired neuronal subtype. For example, Lmx1a in human neural progenitors promotes DA neuron differentiation[9]. However, the above enrichment and selection methods require genetic modification of the cells and therefore are less likely to be clinically applied.
An additional promising application of stem cells is their utilization for drug discovery, and screening of compounds for potential differentiation, survival, therapeutic, teratogenic or toxic effects. Both human ESCs and iPS cells may be utilized to model diseases. For example, human ESCs derived from preimplantation diagnosed affected embryos are used to model the inherited disease that they carry. Human ESCs may be genetically modified to model genetic disorders. Human iPS cells may be derived from patients with both inherited diseases as well as disease of unknown/multifactorial etiology. Differentiated neurons derived from theses pluripotent stem cell models may serve to model the pathogenesis of neural disorders and for the development of new drugs. However, the neuronal cells that are obtained after spontaneous or induced differentiation of pluripotent stem cells are contaminated by undifferentiated stem cells, non-neural cells, neural precursors and glia cells. These contaminating cells interfere with the utilization of the neurons for studying the pathogenesis of neural disorders, drug discovery, including high throughput screening of compounds for neuroprotective and/or therapeutic effect.
An alternative source for neurons may be transdifferentiation of somatic cells by forced expression of transcription factors. Fibroblasts could be converted by this approach to various types of neuronal cells[33].
Neurons that are generated through transdifferentiation may be utilized for multiple applications including toxicology, drug discovery, basic research and cell therapy. Nevertheless, the neurons that are obtained following transdifferentiation are mixed with non-neuronal cells such as the somatic cells of origin and others. The presence of contaminating non-neuronal cells interferes with the use of the transdifferentiated neurons for the various applications.